Antiviral Activity of Tumor Necrosis Factor (TNF) Is Mediated via p55 and p75 TNF Receptors

Mice bearing genetic mutations in TNFR1/p55 (20) and TNFR2/p75 (21) have been previously described. Double TNFR-deficient mice were generated by the appropriate intercrosses of p55 and p75 mutant mice (22, 23). All TNFR-deficient mice used in these studies were hybrids between C57BL/6 and 129/Sv (B6 × 129). Control mice were (B6 × 129)F2. The derivation of p75^−/− and p55^−/−p75^−/− mice has been described elsewhere (21–23). All mice were bred and housed under specific pathogen-free conditions at the Animal Breeding Establishment, John Curtin School of Medical Research (Canberra, Australia). Genotypes of the mutant and wild-type mice were confirmed by PCR. Mice used in these studies were between 6 and 12 wk of age. Previous data show that both strains contribute to resistance to ectromelia virus (EV) and that B6 × 129 are more resistant than either parent (Ruby, J., unpublished results). Both B6 and 129 strains also contribute to resistance to VV and increased resistance to VV is seen in B6 × 129.

The construction of the recombinant vaccinia viruses, VV-HA-TNF and VV-HA-TK (herpes simplex virus thymidine kinase gene), has been previously reported (5, 24). Both viruses also encode the hemagglutinin (HA) gene from influenza virus (A/PR-8). Stocks of rVV were prepared as crude stocks from CV-1 cells. EV (Moscow strain) was prepared as a crude stock from BSC-1 cells.

To study replication of VV-HA-TNF or VV-HA-TK, female mice were inoculated intravenously with 10⁷ PFU of rVV in 200 μl saline. After 3 d, mice were killed and ovaries were harvested for titration of virus using a plaque assay on 143B cells (25). Mice treated with soluble (s)TNFR received a fusion protein of human TNFR2/ p75 linked to the Fc portion of human IgG1 (26). Mice were inoculated intraperitoneally with 300 μg sTNFR on days 0, 1, and 2 after infection and with VV-HA-TNF on day 0. Mice were killed and the ovaries collected on day 3. To investigate EV replication, male mice were infected in the right hind footpad with 5 × 10³ PFU of EV in a volume of 20 μl using a 30 gauge needle. Mice infected with EV were killed 7 d after infection and liver, spleen, and inoculated foot (amputated at the first joint) were harvested. EV was titrated using BSC-1 cells (27). The limit of sensitivity of the infectious plaque assay is 100 PFU. Values below the level of plaque assay sensitivity were assigned a value of 50 PFU.

It has been previously shown that overexpression of TNF by an rVV caused an attenuated infection in mice (5). To determine whether the enhanced clearance of rVV encoding the gene for murine TNF (VV-HA-TNF) was dependent on p55, mice deficient for this receptor were infected with VV-HA-TNF. In three separate experiments, a small but significant (P <0.05) increase in VV-HA-TNF replication was measured in p55-mutant compared to wild-type mice (Table 1). However, the increased replication of VV-HA-TNF in p55^−/− mice represented only partial reversal of attenuation since the yield of the control virus construct, VV-HA-TK, in these mice was three orders of magnitude higher (Table 1). The finding that VV-HA-TNF replication was limited in p55^−/− mice was surprising given that p55 has been shown to mediate the antiviral activity of TNF in vitro (18). Despite the lack of p55, TNF was able to activate antiviral mechanisms which resulted in the rapid resolution of VV infection. The attenuation of VV in this model was dependent on TNF since treatment of p55-mutant mice with sTNFR resulted in significantly increased replication of VV-HA-TNF (P <0.05; Table 1). Thus, the TNF-dependent inhibition of VV-HA-TNF replication was not solely mediated by p55.

To determine the relative roles of p55 and p75 in the attenuation of VV-HA-TNF, mice bearing mutations in either or both TNFRs were infected with VV-HA-TNF. The yields of virus recovered from TNFR-deficient mice indicated that TNF bound both p55 and p75 to initiate antiviral mechanisms (Fig. 1). Although the levels of virus were undetectable in the ovaries of wild-type mice 3 d after infection with VV-HA-TNF, elevated replication occurred in either p55- or p75-mutant mice (P <0.001). The contribution of both receptors to the attenuation of VV-HA-TNF was confirmed by the increase in virus replication in mice deficient for both TNFRs compared to wild-type mice (P <0.01). Indeed, the attenuation of VV-HA-TNF was completely abrogated in the double TNFR^−/− mice, as the rVV replicated to the same extent as the control virus, VV-HA-TK (Table 1 and Fig. 1). There is no evidence from these experiments to suggest that the antiviral activity of TNF was mediated by mechanisms other than via p55 or p75. These data indicate that both p55 and p75 are necessary and sufficient to limit the replication of VV-HA-TNF. In addition, the replication of VV-HA-TNF was significantly greater in p75^−/− and p55^−/−p75^−/− mice compared to p55^−/− mice (P <0.05; Fig. 1).

As described above, the overexpression of TNF could affect antiviral activity. Therefore, we sought evidence of a role for TNF in the physiological control of virus infection. Various strains of TNFR mutant mice were infected with the natural murine pathogen EV. The mice used in these studies were genetically resistant to EV; both C57BL and 129 backgrounds contribute to resistance to EV and B6 × 129 mice were more resistant than either parent strain (Ruby, J., unpublished data, and Karupiah, G., personal communication). Consistent with the resistant phenotype, no virus was detected in the livers of wild-type mice 7 d after infection (Fig. 2). In contrast, virus was demonstrated in the livers of mice with mutations in either TNFR. The level of EV recovered from the livers of p55^−/− mice was consistently higher than that in wild-type mice, although the difference was not statistically significant. However, significantly higher yields of EV were obtained from the livers of p75^−/− (P <0.001) and p55^−/−p75^−/− mice (P <0.01) relative to wild-type mice (Fig. 2). Furthermore, EV replicated to a significantly greater extent in the livers of p75^−/− mice compared to p55^−/− mice (P <0.01; Fig. 2).

Increased levels of virus replication were also measured in the spleens of TNFR mutant mice, compared to control mice, 7 d after infection (Fig. 2). In this organ, significantly higher EV titers were measured in p55^−/− (P <0.05), p75^−/− (P <0.001), and p55^−/−p75^−/− mice (P <0.01) compared to wild-type mice. Very high titers of virus were obtained from the inoculated feet of all strains of mice, consistent with the persistence of EV at this site (28, 29). However, TNF did play a noticeable role in the control of virus replication at this site with significantly higher replication in p55^−/− mice (P <0.05) and each of the p75-deficient strains (P <0.01; Fig. 2). Observations of morbidity of the TNFR mutant mice reflected the yields of virus recovered. The most dramatic effects were seen in p55^−/− p75^−/− mice which showed signs of severe morbidity (ruffled coat, hunched appearance) 2 d after infection. At the time of harvest, the popliteal lymph node draining the inoculation site remained intact only in the control mice. Thus, defects in either TNFR introduced susceptibility to EV.

To further test the antiviral role of the two TNFRs, survival of TNFR mutant mice infected with EV was monitored. Wild-type mice recovered from infection with high doses (5 × 10³ PFU) of the virulent Moscow strain of EV. Prominent swelling of the inoculated footpad was observed in the wild-type mice. However, the footpad resumed a normal appearance ∼14 d after infection. There were no other visible signs of morbidity in these mice. In contrast, clinical signs of susceptibility to EV, ranging from atrophy of the infected foot to skin lesions and even mortality, were seen in the TNFR mutant mice. Like the wild-type mice, all p55^−/− mice survived infection with EV (Fig. 3). However, the reduced resistance of these mice was indicated by the symptoms of mousepox observed. Within 10 d of infection, the inoculated foot of all p55^−/− mice was severely atrophied. Tail lesions were apparent in some (20%) mice after 17 d, but these were resolved at the termination of the experiment (42 d after infection). Mice lacking p75 were significantly more susceptible to EV, with most (80%) mice succumbing to lethal infection in the period from 10 to 21 d after infection (Fig. 3). Morbidity and mortality of p55^−/− p75^−/− mice was consistent with the increased susceptibility of p75^−/− mice. EV infection was lethal for the majority (60%) of p55^−/−p75^−/− mice, which died between 10 and 17 d after infection (Fig. 3). Atrophy of the inoculated hind foot was evident in all mice within 8 d of infection. At the termination of the experiment, the surviving mice showed generalized skin lesions.

Using two different systems of virus infection of mice, we have demonstrated that both p55 and p75 TNFR mediate the antiviral function of TNF in vivo. In an experimental model, the overexpression of TNF during infection with VV was previously shown to lead to rapid elimination of the virus (5). This effect was partially dependent on p55 since the attenuation of VV was reversed to some extent in p55^−/− mice. However, a component of the antiviral mechanism was independent of p55 since treatment of these mice with sTNFR caused further reversal of the attenuation. The antiviral effects of TNF were totally inhibited in mice lacking both TNFRs; in these mice VV-HA-TNF replicated to the same extent as the control virus. Hence, both p55 and p75 were required to activate TNF-dependent antiviral activity. This conclusion was corroborated by studies of the natural murine pathogen, EV, in TNFR^−/− mice. The capacity of TNFR mutant mice to resolve a normally self-limiting infection was impaired. The increased replication of EV in the target organs (liver and spleen) of p55^−/− mice and the clinical symptoms and morbidity consistent with mousepox indicated that p55 plays a role in resistance to this virus. However, the importance of p75 in the antiviral response was highlighted by the increased susceptibility of p55^−/−p75^−/− mice; the lack of both TNFRs resulted in high morbidity and mortality in otherwise resistant mice. Together, these studies indicate that both TNFRs are necessary and sufficient to mediate the antiviral effects of TNF.

Previously, a normal immune response to VV or LCMV was reported (19) in the TNFR1/p55^−/− mice used in this study. We also found that the replication of the control VV construct was not increased in p55^−/− mice (Table 1). VV exhibits only low virulence in mice, and lack of IFN-γ or IFN-α/β, or loss of function of CD8 T cells, is the only factor shown to increase virulence of wild-type VV in resistant mice (25, 27, 30, 31). Consistent with a less important role for TNF in the host response to VV, the sTNFR homologue is truncated in the VV genome (32). Based on the data from this and other studies, it is possible to compare the relative importance of antiviral cytokines. For instance, while TNF clearly plays a determining role in resistance to EV, it is less critical than IFN-γ. When resistant B6 mice were infected with a similar dose of EV, 100% of mice treated with neutralizing anti–IFN-γ antibodies died within 7 d of infection (27). Although EV was lethal for 60–80% of mice deficient for both TNFRs, the survival period was greatly extended, to ∼20 d.

The majority of TNF-dependent functions have been attributed to signal transduction via TNFR1/p55 (17). In contrast, a minor or accessory role has been proposed for TNFR2/p75, with many studies supporting a ligand-passing role for p75 (19, 33, 34). In this scenario, p75 acts to lower the effective concentration of TNF necessary to activate p55 by binding TNF and passing it to p75. However, a distinct role for p75 in mediating cytotoxicity has been suggested in addition to an accessory role (34). In particular, TNF-dependent activation-induced cell death of T cells may be triggered by p75 (21, 35). The use of TNFR-specific antibodies has suggested that both receptors were required for the cutaneous inflammatory response after intradermal injection of TNF (36) and that cytotoxicity was greatly enhanced when both TNFRs were activated (37). These cases suggest that cooperative signaling of TNFR may occur independently of a ligand-passing mechanism. The formation of p55/p75 heterocomplexes has been proposed as an alternative mechanism to describe cooperative signaling between TNFRs (38). In TNF-treated cells, TNFR-specific mAb coprecipitated both p55 and p75. This finding suggests a possible mechanism for the interaction of signaling molecules downstream of either receptor.

A constant observation in mice infected with VV-HA-TNF or EV was that the defect in the antiviral response of p75^−/− mice was greater than that of p55^−/− mice. Although it is well established that the soluble 17-kD form of TNF binds p55 with higher affinity, it has recently been proposed that the membrane-bound 26-kD TNF binds predominantly to p75 (39). It is possible that the dominant role played by p75 in the antiviral studies we report reflects an important role for membrane-bound TNF in activating antiviral activity. In this regard, TNF expressed from cells infected with VV-HA-TNF remained cell-associated 24 h after infection and only reached maximal levels in the supernatant after 48 h (5), when lysis of the infected cells occurred. This contrasts with maximal supernatant levels of secreted cytokines, e.g., IL-2 from similar VV constructs within 12 h of infection (24). Furthermore, the impaired response of p55^−/−p75^−/− mice to virus infection was not different from that of p75^−/− mice. Thus, there was no evidence of synergistic or cooperative interaction between the two TNFRs to support a ligand-passing role for p75 or a role for p55– p75 complex formation in signaling antiviral activity. These findings suggest that p75 plays an independent role in signaling antiviral activity and that p75 appears to be more important than p55 in the control of poxvirus infection. We are currently attempting to address the question of whether different antiviral mechanisms are mediated via each of the TNFRs.

This study was supported by grants from the World Health Organisation Transdisease Vaccinology Program and the Commonwealth AIDS Research Grants Committee. J. Ruby is currently a C.R. Roper Fellow.